Original Article doi: 10.5146/tjpath.2017.01412

Changes in the Hematopoietic System and Blood Under the Influence of Heavy Metal Salts Can Be Reduced with Vitamin E

Anatolii Romaniuk, Mykola Lyndin, Yulia Lуndіna, Vladyslav Sikora, Natalia Hrintsova, Olena Timakova, Olena Gudymenko, Oksana Gladchenko Department of Pathology, Sumy State University, Sumy, Ukraine

ABSTRACT

Objective: The aim of our work was to study the blood parameters and morphological changes in rats exposed to increased amounts of heavy metal salts and the effect of vitamin E. Material and Method: Investigation of bone marrow structural features and blood parameters was performed in sexually mature Wistar male rats (n=84). Results: Exposure to increased amounts of heavy metal salts led to the inhibition of and leukopoiesis, as well as a synchronized increase in the number of which was clearly reflected in the blood: the number of erythrocytes, leukocytes and Hb decreased, and the number of increased. These changes in the blood and bone marrow were less pronounced when vitamin E was used as an adjuster. Conclusion: When increased amounts of HMS enter the rats` bodies, suppression of erythropoiesis and leukocytopoiesis occurs while thrombocytopoiesis increases. These changes depend on the period of intake of heavy metal salts. The adjustment of vitamin E reduces the severity of the cytotoxic effect of heavy metals and improves readaptation in the recovery period. Key Words: Heavy metal salts, Vitamin E, Hematopoietic system, Bone marrow

INTRODUCTION competitive replacement of essential trace elements in the structure of hydroxyapatite, deactivation of enzyme Nowadays hemato-ecological problems are becoming considerably urgent and research is being conducted in systems, DNA damage and so on (6). All these negative this area considering the impact of exogenous factors on impacts can occur in the process of hematopoiesis, and this biocoenosis. Some of the most powerful pollutants are is reflected in the peripheral blood. heavy metal salts (HMS), and in some districts of the Recently more attention has been paid to the investigation of Sumy region of Ukraine, their concentrations exceed the changes in hematopoietic tissue. This has been achieved by maximum permissible range in the water and soil (1). means of trephine biopsy of the iliac bone and preparation Unfortunately, relatively little research has been devoted to of histological specimens that allow evaluation of the the study of the impact of heavy metal compounds (HMC) parenchymal component of bone marrow and changes in on the hematopoietic system despite the rather clear impact the stroma, which play a regulatory role in the proliferation of these factors on the majority of organs (2). Moreover, the and maturation of hematocytes (7). research available on this theme is devoted only to blood, or only to the hematopoietic system in most cases (3,4). Another important objective of modern medicine is the search of adequate corrective and preventive ways that Qualitative and quantitative blood indicators reflect the increase body resistance to the conditions of constantly function of the hematopoietic system and the operation of growing urbanization and technological progress that lead other internal organs. The influence of pathogenic factors to rapid contamination of the environment. related to HMS on the process of hematopoiesis may occur through their direct impact on the bone marrow (BM) or The purpose of our work was the study of the laboratory indirect damage of other internal organs (5). Their negative parameters of blood and morphological changes of bone effects have been listed as lipid peroxidation induction, marrow in rats exposed to increased amounts of HMS

Correspondence: Anatolii Romaniuk Sumy State University, Department of Pathology, Sumy, Ukraine Received : 30.05.2017 Accepted : 18.07.2017 E-mail: [email protected] Phone: +38 097 715 55 24

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to investigate the possible correction of the changes with Blood obtained from the rats` aorta served as the material vitamin E. for laboratory research in which the number of erythrocytes, leukocytes, platelets, hemoglobin (Hb), erythrocyte MATERIAL and METHODS sedimentation rate, and the levels of Ca, Na, K, creatinine The investigation of bone marrow structural features and and urea were determined when the rats were taken out of blood parameters was performed in sexually mature Wistar the experiment. male rats (4 months old, n=84), considering the impact of the animals` ages on the process of hematopoiesis. All The study of structural bone marrow peculiarities was investigations were performed in accordance with “General performed using the femur. The material was fixed in ethical animal experimentation” (Kyiv, 2001) and the 10% buffered formalin solution. Decalcification had Helsinki Declaration of the General Assembly of the World been conducted with EDTA solution (pH 7.0) for 14 Medical Association (2000). Rats were kept on a standard days, with daily change of the solution. After standard diet (except some peculiarities in water consumption) in tissue processing, 4μ-thick sections were taken from the vivarium at 20-25ºC temperature, no more than 50% paraffin-embedded tissues and stained for H&E. In order humidity, and at day/night light mode. to differentiate erythropoietic and leukopoietic lines of hematopoiesis, immunohistochemical studies were The distribution of the animals into the groups was as performed to determine the receptors to myeloperoxidase, follows: CD3 and CD79α. Unfortunately, cytological examination The first group included three series of rats (12 rats in each and quantitative assessment of the stromal component series). The first series was used as control and the animals could not be performed due to the technical difficulties. drank ordinary water. In the second series, the rats received The measurement of the size of microspecimen constituent aqueous mixture of heavy metal salts in the concentration elements was conducted in the environment of the appropriate for the Sumy region and containing 5 mg/l zinc «Digimizer» morphometric program (Figure 1). Obtaining (ZnSO x7H O), 1 mg/l copper (CuSO x5H O), 10 mg/l 4 2 4 2 and storage of the images was carried out by means of iron (FeSO ), 0.1 mg/l manganese (MnSO x5H O), 0.1 4 4 2 the digital image output system «SEO Scan» (Ukraine). mg/l lead (Pb(NO ) ), and 0.1 mg/l chromium (K Cr O ) 3 2 2 2 7 Statistical calculations were performed using Microsoft (1). In the third series, the rats received the same heavy Excel 2010 and Attestat 12.0.5. Non-parametric tests metal nsalts mixture with vitamin E correction (9.1 mg/kg were performed with the Mann-Whitney U test while the of 10% oil oral solution). The animals` dosage calculation Spearman correlation test was used in correlation analyses. was based on the average daily therapeutic dose for adults. The results were considered statistically reliable at a To examine the effects of subacute and chronic exposure to probability level of more than 95% (p <0.05). HMS on the blood and hematopoiesis, 6 animals in each series of the first group were removed from the experiment RESULTS by decapitation under ether anesthesia on the 30th and Both upwards and downwards variations in blood 90th days of the study. test indicators were found (Figure 2), establishing the The second group, including four series of laboratory dynamic changes in blood under the influence of HMS animals with 12 rats in each series, was used to investigate and their adjustment with vitamin E, as the duration of the readaptation process. The first series was control. The the experiment was prolonged. Blood counts showed second included rats that had been consuming a mixture the decrease in erythrocytes, leukocytes and Hb, and the of heavy metal salts solution for 90 days and then moved increase of platelets while rats were receiving HMS. to ordinary drinking water. In the third series, the rats had been consuming a mixture of heavy metal salts solution with After day 30, Hb decreased by 11% (p<0.01) and the vitamin E correction for 90 days and had then been moved number of erythrocytes by 23.3% (p<0.01) in rats of the to ordinary drinking water and continued to use vitamin second series of the first group. There was a 4.6% increase in E. In the fourth series, the animals had been consuming a the number of leukocytes and 2.5% increase in the number mixture of heavy metal salts solution for 90 days and had of platelets but were not found to be significant (p=0.11 and then started to use ordinary drinking water with vitamin E. p=0.08, respectively). With prolonged duration of receiving The process of rapid and remote readaptation was examined exogenous pollutants (on day 90), a consistent decrease in by taking 6 animals in each series out of the experiment on Hb levels and in the number of leukocytes and erythrocytes the 30th and 90th days. was observed (18.4%, 32.5%, 15.3%, respectively) while

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platelets were increased by 11% (p<0.01). The changes were 6.6%, the number of erythrocytes by 23.3% and leukocytes not so impressive in the blood of rats in the third series of by 13.2%, while the number of platelets conversely increased the first group. On the 90th day, Hb indicators decreased by by 8.2% (p<0.01).

Figure 1: A screen shot as an example of working with images in the environment of the «Digimizer» morphometric program.

Figure 2: Indicators of rats’ complete blood counts. Exp: the period of the experiment, Read: the period of readaptation. Blue line: control group, red line: rats that drank water with HMS, green line: rats that drank water with HMS and were administered vitamin E, violet line: rats that were administered vitamin E during rehabilitation after HMS.

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In the readaptation process, blood indicators gradually decrease in the number of erythropoietic and leukopoietic normalized, but did not reach the levels of animals in the cells with simultaneous increase in the number of control group. The tempo of this process also depended megakaryocytes was taking place (control group indicators on the conditions of readaptation. Thus, in rats of the were 5.25±1.42%, 14.75±1.42%, 0.16±0.05% at the second series of the second group, increases in Hb levels epiphysis level and 17.83±0.75%, 51.17±3.27%, 0.16±0.05% and erythrocyte and leukocyte numbers were observed at the diaphysis level, respectively (8)), which reached (12.6%, 35.9%, 8.4%, respectively), while numbers maximum values on the 90th day of the experiment. Thus, decreased by 5.3% (p<0.01) on the 90th day. Increases in Hb the area of erythropoiesis in the second series of rats was levels and erythrocyte and leukocyte numbers were 17.5%, reduced by 30% at the epiphysis level (p=0.03) and by 39.3%, 11.5%, respectively, in animals of the fourth series 17.8% at the diaphysis level (p=0.025) in 3 months; the of the second group; and 8.9%, 26.3%, 11.3%, respectively, area of leukopoiesis decreased by 6.1% (p=0.16) and 13.7% in rats of the third series of the second group. For these last (p<0.01), respectively. The number of megakaryocytes two groups, decreases in platelet numbers were found at increased by 27%. rates of 6.8% (p<0.01) and 5.6% (p<0.01), respectively. Adipose tissue and sinusoids crowded with blood replaced The changes in blood biochemical parameters in the rats the area being released from hematopoietic tissue during of the second and third series of the first group had similar the experiment (normally they accounted for 11±2% and dynamics (Table I), which was expressed by decreases in 18.7±2.1%, respectively) (8). the measured values of Ca (p<0.01) and K (p=0.01) and Hematopoiesis indicators gradually returned to normal by increases of Na (p<0.01), creatinine (p=0.04) and urea during readaptation, but they did not reach the values of (p<0.01). During readaptation, these indicators gradually the control group of animals (Figure 5). The speed and the normalized, but in most cases they did not reach the values completeness of recovery depended on the use of vitamin E of animals in the control group. The speed of recovery for adjustment. In rats of the second series, erythropoiesis depended on readaptation conditions (whether adjustment increased by 35.6% and 18.1% (p=0.01) respectively in the with vitamin E was present or absent) and readaptation femur segment, leukopoiesis increased by 9.5% (p=0.045) duration. and 8.4 % (p=0.01), and thrombopoiesis decreased by 18% In the study of morphological features of bone marrow on the 90th day of readaptation. In animals of the third structure, we found that it undergoes quantitative and series, the area occupied by erythropoiesis increased by qualitative changes during the experiment, both at the level 29.5% (p=0.01) at the epiphysis level and by 18.5% (p<0.01) of the epiphysis and the diaphysis (Figure 3,4). A gradual at the diaphysis level. Leukopoietic areas increased by 5.8%

Table I: Indicators of the biochemical blood tests of the animals. Group I Group ІІ Series 1 Series 2 Series 3 Series 1 Series 2 Series 3 Series 4 30th 90th 30th 90th 30th 90th 30th 90th 30th 90th 30th 90th 30th 90th day day day day day day day day day day day day day day 3.03 3.0 2.75** 2.81* 2.87* 2.81** 3.03 3.01 2.88 2.94 2.84 2.98** 2.87 2.96* Ca ±0.06 ±0.07 ±0.05 ±0.11 ±0.12 ±0.05 ±0.08 ±0.09 ±0.05 ±0.13 ±0.12 ±0.05 ±0.07 ±0.08 134.9 135.3 144.6** 155** 140.1* 147.5** 135.1 135.6 149.6* 143.8** 142.3 137** 146.3** 139.9** Na ±4.1 ±3 ±2.6 ±3.6 ±1.1 ±3.1 ±1.6 ±2.5 ±2.3 ±4.4 ±1.5 ±2.6 ±2.6 ±2.5 4.68 4.7 4.3* 3.98** 4.5 4.2* 4.65 4.68 4.17* 4.37** 4.3 4.55** 4.2 4.45** К ±0.32 ±0.33 ±0.14 ±0.15 ±0.23 ±0.1 ±0.27 ±0.17 ±0.1 ±0.12 ±0.14 ±0.16 ±0.18 ±0.23 78.7 79.1 88.3** 90.4** 84.8* 87.1* 78.5 78.9 86.8** 82.4** 83.1* 80.1** 84.5** 81.7** Cr ±4.9 ±5.6 ±2.3 ±1.2 ±3.3 ±1.7 ±2.7 ±2.4 ±1.9 ±1.8 ±3.1 ±1.3 ±1.9 ±1.6 4.89 4.87 5.73** 6.12** 5.38* 5.73** 4.85 4.83 5.63 5.15* 5.28** 4.95** 5.47* 5.28* U ±0.35 ±0.21 ±0.52 ±0.46 ±0.29 ±0.19 ±0.19 ±0.28 ±0.26 ±0.38 ±0.15 ±0.22 ±0.45 ±0.4 Ca: Calcium; Na: Sodium; K: Potassium; Cr: Ceatinine, U: Urea. * The probability value (p) between two independent samples according to the U criterion on the basis of the Mann-Whitney criterion p <0.05, ** p <0.01.

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(p=0.03) and 11.4% (p<0.01) respectively, and the number area (r=0.7, p<0.001 and r=0.87, p<0.001, respectively) of platelets decreased by 23%. Indicators of the third series and the platelet levels and the number of megakaryocytes of animals were as follows: erythropoiesis increased by (r=0.47, p<0.001). Hb values and their sensitivity were 35.5% and 18.9% (p=0.01) and leukopoiesis by 10.5% identical to erythrocytes: as the erythropoiesis area became (p=0.03) and 9.4% (p=0.01), and the number of platelets smaller, the rates of Hb decreased (r=0.47, p<0.001 and decreased by 36%. The area occupied by predecessors of T- r=0.36, p=0.002 in the appropriate topography of the and B- (CD3 and CD79α receptors expression) femur). did not show any statistically significant changes (p>0.05) Among the qualitative changes in rats’ bone marrows during the experiment and readaptation and it was about during the experiment, we observed hemorrhages, focal 5%. adiposis (due to inhibition of hematopoiesis), myxomatosis Comparing the area occupied by leukocyte predecessors phenomena, sinusoidal ectasia, histiocytic infiltration, to the area of erythropoiesis (the myeloid/erythroid (M:E) degenerative changes (atrophy, necrosis and apoptosis ratio), a gradual increase was established (due to more rapid phenomena) and others (Figure 6). The intensity of these suppression of erythropoiesis) from 1:2.8 (normal) to 1:3.46 changes and the rate of their recovery depended on the (in the 90 days of the experiment). During readaptation, we adjustor availability - changes developed slowly and not in have observed some optimization of the M:E ratio (1:2.75), full extent when using vitamin E. but it did reach normal values due to the lack of complete DISCUSSION recovery of hematopoiesis. The bone marrow undergoes significant changes during Laboratory and morphological parameters of the blood- vital activity, which is associated with age characteristics forming system were monitored during the whole period and the impact of the environment (4,9). The study was and we established a direct correlation between the number carried out on 84 rats to determine the impact of increased of erythrocytes and erythropoiesis area (r=0.71, p<0.001 for amounts of HMS on the hematopoietic system. We found the epiphysis and r=0.67, p<0.001 for the diaphysis), as well that exposure to exogenous pollutants led to the inhibition as between the number of leukocytes and the leukopoietic

Figure 3: Indicators of bone marrow area that are engaged in various pathways of hematopoiesis in rats. Exp: The period of the experiment, Read: The period of readaptation, C: indicators in the control series of rats, Blue Line: Rats that drank water with HMS, Red Line: Rats that drank water with HMS and were administered vitamin E, Green Line: Rats that were administered vitamin E during rehabilitation after HMS.

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of erythropoiesis and leukopoiesis and synchronized presence of estrogen receptors in hematopoietic cells (11). growth in the number of megakaryocytes which was We do not rule out a direct cytotoxic effect of metals on clearly reflected in the blood: the number of erythrocytes, blood corpuscles (3). Reactive thrombocytosis is also leukocytes and Hb decreased, and the number of platelets associated with slowly progressive anemia, and as a result, increased. These changes are associated with the direct and the rate of maturation of platelet predecessors increases and indirect hematotoxic influence of HM, directly affecting their destruction decreases (12). The study showed more the function of hematopoietic cells (increased lipid pronounced changes in hematopoiesis - the blood system, peroxidation and the formation of active oxygen forms, the namely during the subacute period of the experiment DNA destabilization, deactivation of enzyme systems) (6). (30 days) with a gradual slowing on the 90th day, which HM suppresses the proliferative activity of cells and blocks is associated with activation of endogenous adaptive their maturation. The estrogen-resembling activity of HM compensatory processes in rats when their living conditions plays a significant role in the inhibition of hematopoiesis change. (10), which is reflected in a normal blood test (erythrocytes As seen from the results of the study, significant changes and Hb indicators of men are higher and platelet indicators in biochemical blood tests (increased urea, creatinine, are lower compared to women). Therefore, the increase in dismicroelementosis) occur due to the nephrotoxic effects the number of HM in the blood enhances their estrogen- of HMS. There are also negative effects on bone tissue resembling effect on the bone marrow, considering the

1A 2A 3A

1B 2B 3B

Figure 4: Longitudinal section of rat femur on diaphysis. 1) Control group, 2) Rats of the second series of the first group (30th day), 3) Rats of the second series of the first group (90th day). (A: H&E; x400, B: Immunohistochemical determination of myeloperoxidase receptors; ×400).

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and the parathyroid gland (initial decrease of Ca because As HMS strongly inducts lipid peroxidation, we used of probable inhibition of parathormone synthesis and vitamin E as an adjustment agent that has a strong protective subsequent increase in the washout of calcium from the effect on lipids and NAD as a common antioxidant (15). We hydroxyapatite bone structure) (13,14). Indirect effects of observed the protective effects of vitamin E, as the reparative HMS on hematopoiesis are realized precisely because of changes occurred more fully in vitamin E administered rats. increasing metabolic products of nitrogenous bases and It is also stated that vitamin E does not have any significant dismicroelementosis. negative effect on the process of hematopoiesis in mature rats (16). However, the changes that occurred during the Erythropoietic tissue was found to be the most sensitive experiment were not completely eliminated in any case part of bone marrow. Its inhibition reaches 36.6% even with the constant administration of vitamin E. This (leukopoiesis is maximally inhibited by 14%), which finding emphasizes the cumulative properties of exogenous manifests itself as a rapidly arising anemia. Anemia can pollutants. Also, we have not observed any significant stimulate thrombocytosis, which is also provoked by heavy difference in the hematopoiesis recovery rate during metals. It seems that the least affected part of hematopoiesis different periods of rehabilitation, indicating the gradual is . Other studies are needed to see whether and slow excretion of HMS. lymphoid tissue is affected in the other parts of the lymphoid system, i.e. the thymus, spleen, lymph nodes, etc.

4A 5A 1A

4B 5B 1B

Figure 5: Longitudinal section of rat femur on the diaphysis. 1) Control group, 4) Rats of the second series of the first group (30th day), 5) Rats of the second series of the first group (90th day). (A: H&E; x400, B: Immunohistochemical determination of myeloperoxidase receptors; x400).

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1 2 3

4 5 6

7 8 9

A B C

Figure 6: Qualitative changes in rats’ bone marrow influenced by HMS. 1) Focal lipomatosis (H&E; x100). 2) Clusters of megakaryocytes (H&E; x100). 3) Sinusoidal ectasia (H&E; x100). 4) Myxomatosis (H&E; x100). 5) Atrophy (H&E; x100). 6) Hemorrhages (H&E; x100). 7) Emperipolesis (H&E; x400). 8) Dysmegakaryopoiesis (H&E; x400). 9) Apoptosis (H&E; x400). (A: with a leukocyte inside, B: Wrinkled megakaryocyte with symptoms of kariopiknosis and chromatin condensation, C: Cells in the state of apoptosis)

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In conclusion, the bone marrow is very vulnerable and also 5. Travlos GS. Histopathology of bone marrow. Toxicol Pathol. very sensitive to the variability of living conditions, which 2006;34:566-98. leads to both a parenchymal and stromal reaction. Increased 6. Jaishankar M, Tseten T, Anbalagan N, Mathew BB, Beeregowda amounts of heavy metal salts oppress erythropoiesis and KN. Toxicity, mechanism and health effects of some heavy metals. Interdiscip Toxicol. 2014;7:60-72. leukocytopoiesis in rats while thrombotcytopoiesis occurs, as demonstrated in our study. The level of qualitative and 7. Frenkel MA, Chigrinova EV, Kupryshina NA, Pavlovskaia AI. Diagnostic value of study of bone marrow trepanobiopsy quantitative destructive changes depends on the direct and imprints in patients with non-Hodgkin’s lymphomas. Klin Lab indirect effects of exogenous pollutants. More pronounced Diagn. 2007;11:44-7. changes were observed during the subacute period of the 8. romaniuk A, Lyndina Yu, Sikora V, Lyndin M, Karpenko L, experiment (first 30 days) with gradual slowing on the 90th Gladchenko O, Masalitin I. Structural features of bone marrow. day, which is associated with activation of endogenous Interv Med Appl Sci. 2016;8:121-6. adaptive-compensatory processes in rats when living 9. Cline JM, Maronpot RR. Variations in the histologic distribution conditions change. of rat bone marrow cells with respect to age and anatomic site. Toxicol Pathol. 1985;13:349-55. The adjustment of vitamin E reduces the severity of the 10. Iavicoli I, Fontana L, Bergamaschi A. The effects of metals as cytotoxic effect of heavy metals and improves readaptation endocrine disruptors. J Toxicol Environ Health B Crit Rev. in the recovery period. However, even with constant use of 2009;12:206-23. vitamin E, the full range of morphological changes of the 11. Schulze H, Shivdasani RA. Mechanisms of thrombopoiesis. J bone marrow does not reach normal levels, which reflects Thromb Haemost. 2005;3:1717-24. the cumulative properties of heavy metal salts in the body. 12. koloskov AV, Saparkina MB, Philippova OI, Stolitsa AA. Reactive thrombocytosis. Transfusiology. 2012;13:359-71. CONFLICT of INTEREST 13. Kuzenko Y, Romanyuk A, Korobchanskaya A, Karpenko L. The authors declare no conflict of interest. Periodontal bone response under the influence of Cr(VI). Osteologický Bulletin. 2014;1:23-7. REFERENCES 14. Sabath E, Robles-Osorio ML. Renal health and the environment: 1. yakovtsova AF, Gubina-Vakulik GI, Sibbons P, Gorbatch TV, Heavy metal nephrotoxicity. Nefrologia. 2012;32:279-86. Ansari T, Markovsky VD, Sorokina IV, Potapov SN, Gargin 15. Górnicka M, Drywień M, Frąckiewicz J, Dębski B, Wawrzyniak VV. Morphological and chemical changes in the medulla of A. Alpha-tocopherol may protect hepatocytes against oxidative the adrenal glands of progeny from parents who smoked pre- damage induced by endurance training in growing organisms. conception: An experimental study in rats. Comp Clin Pathol. Adv Clin Exp Med. 2016;25:673-9. 2007;16:131-7. 16. Lucas EA, Chen TY, Chai SC, Devareddy L, Juma S, Wei CI, 2. Romaniuk A, Korobchanska AB, Kuzenko Y, Lyndin M. Tripathi YB, Daggy BP, Hwang DF, Arjmandi BH. Effect of Mechanisms of morphogenetic disorders in the lower jaw under vitamin E on lipid parameters in ovariectomized rats. J Med the influence of heavy metal salts on the body. Interv Med Appl Food. 2006;9:77-83. Sci. 2015;7:49-52. 3. Saljooghi AS, Delavar-Мendi F. The effect of mercury in iron metabolism in rats. J Clinic Toxicol. 2012:3:1-5. 4. Hounkpatin AY, Edorh PA, Guédénon P, Alimba CG, Ogunkanmi A, Dougnon TV, Boni G, Aissi KA, Montcho S, Loko F, Ouazzani N, Mandi L, Boko M, Creppy EE. Haematological evaluation of Wistar rats exposed to chronic doses of cadmium, mercury and combined cadmium and mercury. Afr J Biotechnol. 2013;12:3731- 7.

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